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Graphdiyne Nanosheet-Based Drug Delivery Platform for Photothermal/Chemo-therapy Combination Treatment of Cancer jun jin, Mengyu Guo, Jiaming Liu, Jing Liu, Huige Zhou, Jiayang Li, Liming Wang, Huibiao Liu, Yuliang Li, Yuliang Zhao, and Chunying Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17219 • Publication Date (Web): 19 Feb 2018 Downloaded from http://pubs.acs.org on February 19, 2018

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ACS Applied Materials & Interfaces

Graphdiyne Nanosheet-Based Drug Delivery Platform for Photothermal/Chemo-therapy Combination Treatment of Cancer

Jun Jin1,3, Mengyu Guo1,3, Jiaming Liu1,3, Jing Liu1,3, Huige Zhou1,3, Jiayang Li1,3, Liming Wang1,3, Huibiao Liu2,3*, Yuliang Li2,3, Yuliang Zhao1,3 and Chunying Chen1,3* 1. Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience; National Center for Nanoscience and Technology, University of Chinese Academy of Sciences, Beijing 100190, P.R. China 2. CAS Key Laboratory of Organic Solids, Beijing National Laboratory for Molecular Sciences (BNLMS), CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China 3. University of Chinese Academy of Sciences, Beijing 100049, P. R. China

ABSTRACT Nowadays, two-dimensional (2D) materials have attracted extensive attention as cancer drug delivery platforms, owing to their unparalleled physicochemical properties and superior specific surface area. Graphdiyne (GDY) is a novel 2D carbon material. Compared to graphene, GDY not only has benzene rings composed of sp2 hybridized carbon atoms, but also has acetylene units composed of sp hybridized carbon atoms, therefore it possesses multiple conjugated electronic structures. Herein, we used doxorubicin (DOX) as a model drug to develop a GDY nanosheet-based drug delivery platform for a photothermal/chemo-therapy combination in living mice. With a high photothermal conversion ability and drug loading efficiency, GDY/DOX under an 808 nm laser irradiation showed a much higher cancer inhibition rate compared with solo therapy both in vitro and in vivo. Furthermore, GDY exhibited great biocompatibility and no obvious side effects, as shown by histopathological examination and serum biochemical analysis. For the first time, our work demonstrated a successful example of GDY for efficient photothermal/chemo-therapy and suggests both safety and great promise for GDY in cancer treatment. KEYWORDS: graphdiyne, doxorubicin, drug delivery, photothermal therapy, chemotherapy, combination treatment

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INTRODUCTION Cancer is one of the most difficult diseases to manage in the world.1 Conventional therapy, such as surgery, radiation and chemotherapy, cannot meet the clinical demand, due to poor treatment outcomes, high risks of recurrence, and intolerable side effects. To improve the survival rate of patients suffering from cancer, a novel combination therapy with high efficiency is essential. With the development of nano-biotechnology, nano-structure materials are attracting immense interest for development as cancer theranostic agents.2 Among them, a large family of nano-carbon materials, including mesoporous carbon, carbon nanodots (CNDs), fullerenes, carbon nanotubes (CNTs), graphene and their derivates, has been extensively studied for multimodal nanomedicine due to their unique physicochemical properties.3-5 In the last two decades, the research focus has gradually been changing from zero-dimensional (CNDs) or one-dimensional materials (CNTs) to two-dimensional (2D) materials (graphene). Graphene is a monolayer or a few layers of a honeycomb lattice structure sheet consisting of sp2-hybridized carbon atoms.6 Owing to its structural and geometrical features, graphene exhibits remarkable mechanical, optical, thermal and electrical properties.7-8 Furthermore, graphene has a great capability to immobilize various types of biomolecules, fluorescent probes and drugs through non-covalent and covalent interactions for various applications in medicine, including drug/gene delivery, photothermal/photodynamic therapy, diagnostic imaging, and biosensing.9-12 However, graphene and its derivates suffer from potential toxicity to organism, possibly due to oxidative stress and inflammation.13-15 Graphdiyne (GDY) is a novel 2D carbon material. Compared to graphene, GDY not only has benzene rings composed of sp2 hybridized carbon atoms, but also has acetylene units composed of sp hybridized carbon atoms, therefore it possesses multiple conjugated electronic structures.16 Although the structure of graphdiyne has been theorized in 1968, it is only since 2010 that GDY have been synthesized on a large scale via a homocoupling reaction using hexaethynylbenzene on the surface of copper.17 GDY is a metal-free-semiconductor material, and possesses a tunable bandgap from 0.14 eV to 1.22eV based on the number of layers and manner of stacking.18 It has exhibited tremendous potential in energy field applications.19-21 However, using GDY as a drug delivery platform for cancer treatment has not been investigated up to now. Considering the broad absorption of GDY across the entire visible light region, it can also be utilized as a photothermal treatment (PTT) agent. 21 With these unique properties, GDY has promising prospects as a multi-modal therapy platform for cancer. Herein, we present a GDY-based drug delivery platform for a photothermal/chemotherapy combination treatment for cancer. 2D GDY nanosheets were fabricated by an exfoliation method. We found GDY to have an excellent capability to load doxorubicin (DOX) on the sheet surface mainly through π-π stacking and electrostatic interactions (Scheme 1). With a good photothermal effect, DOX-loaded GDY (GDY/DOX) exhibits a pH-/photo-responsive release behavior. As expected, both in vitro and in vivo, GDY/DOX displays a dramatically enhanced


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ability to kill tumor cells, benefiting from the photothermal/chemotherapy combination.

Scheme 1. Illustration of GDY/DOX and the Combination Treatment of Cancer

EXPERIMENTAL SECTION Materials. Doxorubicin (DOX) was acquired from Beijing HVSF United Chemical Materials CO., Ltd. N-terminal polyethylene glycol (PEG-NH2, average Mw ≈ 2000) was acquired from Beijing JenKem Technology Co., Ltd. Ultrapure Milli-Q water with resistivity of an 18.2 MΩ was employed for all the experiments. Preparation of GDY nanosheets. GDY powder was supplied from Yuliang Li’s group (Chinese Academy of Sciences), which synthesized it according to the previously published procedure.17 The GDY nanosheets were prepared using a common liquid exfoliation technique. Briefly, 5 mg of the GDY powder was mixed with 5 mL of 2 mg/mL PEG-NH2 solution, then sonicated in an ice bath for 4 h. The obtained mixture was centrifuged at 3000 rpm for 10 min to get rid of residual unexfoliated particles and the supernatant was stored at 4 °C. Before use, excess PEG-NH2 in the GDY nanosheets solution was removed through centrifuging at 13000 rpm for 5 min. Characterization of GDY nanosheets. The morphologies were observed using an FEI Tecnai G2 F20 transmission electron microscopy (TEM) instrument. The sizes and heights were measured by a Bruker Multimode-8 atomic force microscope (AFM). Tecan Infinite M200 spectrophotometer was employed to obtain UV-vis-NIR spectra and fluorescence spectra. The polydispersity index (PDI), hydrodynamic diameter (Dh) and Zeta potential were measured using a Malvern Zetasizer instrument. X-ray photoelectron spectroscopy (XPS) was collected on a Thermo Fisher XPS instrument. Photothermal effect of GDY nanosheets. The GDY nanosheets aqueous solutions with different concentration (0, 50, 100, 200 μg mL-1) were irradiated with



an 808 nm laser (2 W cm-2). The laser was provided by a fiber-coupled diode continuous wave laser system, and the temperature variations of GDY were monitored by a compact infrared (IR) thermal imaging camera. Drug loading and release. GDY nanosheets (200 μg/ml) were mixed with various concentrations of DOX (100, 200, 300 or 400 µg/ml) in PBS (pH = 7.4), then stirred vigorously at room temperature shielded from light overnight. The DOX-loaded GDY nanosheet (GDY/DOX) thus obtained was centrifugated and washed with ultrapure water. Afterward, the GDY/DOX were re-dispersed in ultrapure water for further use. The concentration of DOX in the supernatant was measured by UV-vis-NIR spectra at 480 nm to calculate the DOX loading content and entrapment efficiency. Loading content = (weight of DOX in GDY/DOX)/ (weight of GDY/DOX). Entrapment efficiency = (weight of DOX in GDY/DOX)/(initial weight of DOX). For the pH-promoted DOX release, GDY/DOX in PBS at two different pH (5.0 and 7.4) was stirring at 37 °C in a water bath shielded from light. The solution was centrifuged at the desired time points to collect the supernatant. Then, the fresh PBS was added into the residual solution to restore the original volume. The concentration of released DOX in the supernatant was measured by a UV-vis-NIR spectra at 480 nm. The similar procedures were used in the photo-promoted DOX release experiments. The GDY/DOX solutions were irradiated with an 808 nm laser (2 W cm-2). Cell culture assays. The human breast carcinoma cell line MDA-MB-231 and MCF-7 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium (WISENT Inc.). Human lung carcinoma cells A549 and human bronchial epithelial cell 16HBE were cultured in RPMI-1640 medium (WISENT Inc.). All the cell culture media were supplemented with 10% fetal bovine serum (FBS) (Gibco) and 100 U/mL penicillin/streptomycin (Invitrogen). Cells were cultured at 37 °C in a humidified atmosphere with 5% CO2. Intracellular DOX release. The MDA-MB-231 cells were seeded into 6-well microplates at a density of 5 × 105 cells/well. After incubation for 24 h, GDY/DOX was added into the microplates with a final concentration of 5.4 μg/mL. The fluorescent images were taken by a fluorescence microscopy at 0.5, 2, 4, and 6 h post treatment. For the laser-promoting drug release experiment, the cells were cultured with GDY/DOX for 2 h. After that the cells were irradiated with an 808 nm laser (2 W cm-2) for 5 min, followed by culturing for half an hour. Finally, the cells were washed with PBS. The intracellular fluorescence of DOX was quantified using a flow cytometer (FACS-Calibur, Becton Dickinson, USA). Inhibition of tumor cells of GDY-DOX. The cytotoxicity of GDY-DOX in vitro was determined with a Cell Counting Kit-8 (CCK-8) (Dojindo, Japan). The cells were planted in 96-well microplates at a density of 5000 cells/well and incubated for 12 h. Then the culture medium in each well was replaced with fresh complete medium containing 0, 1.08, 2.16, and 5.4 μg/mL GDY and GDY/DOX (equivalent DOX concentration 1, 2, and 5 μM). After 4 h, the cells were irradiated with an 808 nm laser (2 W cm-2) for 5 min. After another 24 h, cell viability was assessed and calculated as the ratio of the absorbance of treated and untreated wells. The


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absorbance at 450 nm and 650 nm (background reference) were measured by an Infinite M200 microplate reader. For PTT therapy, after being cultured for 4 h at the 5 μM DOX equivalent concentration, the cells were irradiated with an 808 nm laser (2 W cm-2) for 5 min. After incubation for 24 h, the medium was replaced with fresh medium containing Calcein-AM (5 g mL-1) and PI (10 g mL-1). Then incubation for half an hour, the cells were rinsed with PBS and imaged by a fluorescence microscopy. Live cells showed a green color while a red color represented for dead ones. In addition, the identically treated cells in a 96 well microplate were used to perform the CCK-8 assay to understand how DOX released by NIR laser irradiation influences cell viability. Antitumor activity evaluation. Tumor-bearing mouse model was developed by subcutaneously injecting 1×106 MDA-MB-231 cells at the right hind leg of BALB/c nude mice. The tumor volume (mm3) = length × width2/2. When the tumor grew to approx. 100 mm3, the mice were randomly separated into eight groups: (a) PBS, (b) PBS + laser, (c) DOX, (d) DOX + laser, (e) GDY, (f) GDY + laser, (g) GDY/DOX, (h) GDY/DOX + laser. For groups (a) and (b), the mice were intravenously injected 200 μL of PBS. For groups (c) and (d), the mice were given an intravenous injection of 200 μL of DOX (0.54 mg/mL, i.e. 5.4 mg Dox/kg of body weight). For groups (e) and (f), the mice were given an intravenous injection of 200 μL of GDY (1 mg/mL, i.e. 10 mg GDY/kg of body weight). For groups (g) and (h) the mice were intravenously administrated 200 μL of GDY/Dox (1.54 mg/mL, with an equivalent Dox dose of 0.54 mg/mL and GDY of 1 mg/mL). The groups (b), (d), (f) and (h) were then irradiated by an 808 nm laser (2 W cm-2, 10 min). During the irradiation process, the local temperature of the tumors was recorded using an IR thermal imaging camera. In the following 3 weeks, the tumor growth was monitored, and then the mice were sacrificed. The tumors were dissected and weighed to evaluate the therapeutic efficacy. The serum samples and main tissues (heart, liver, spleen, lung and kidney) were gathered for the serum biochemical analysis and histopathological examination.

RESULTS AND DISCUSSION Characterization of GDY nanosheets. The TEM and AFM images showed that the average size of GDY nanosheets is about 160 nm (Figure 1a-c). The average height of GDY nanosheets was determined to be about 4.7 nm by AFM (Figure 1c). The hydrodynamic size of GDY nanosheets was determined by DLS to be 212.2 ± 70.4 nm with a polydispersity index of 0.311 (Figure 1d). The hydrodynamic size of GDY nanosheets had no significant change in PBS or FBS during one week, confirming its stability (Figure 1e). The chemical composition of GDY nanosheets was assessed with XPS. As shown in Figure S1, GDY nanosheets are composed only of elemental carbon. The presence of an O signal is due to the absorption of air on the GDY nanosheets. The high resolution asymmetric C 1s XPS of GDY shows four characteristic sub-peaks representing for sp2 (C=C) at 284.5 eV, sp (C≡C) at 285.2 eV, C-O at 286.9 eV, and C=O at 288.5 eV (Figure 1f). The area ratio of the sp (C≡C)/sp2 (C=C) units was



about 2, which is consistent of the expected chemical composition of GDY nanosheets.

Figure 1. (a) TEM image of GDY nanosheets, (b) AFM image of GDY nanosheets, (c) thickness of GDY nanosheets measured by AFM, (d) size distribution of GDY nanosheets in water measured by DLS, (e) stability of GDY nanosheets in PBS or FBS during a week, (f) XPS spectrum of GDY nanosheets.

Considering that GDY nanosheets have an absorption covering the whole near IR, it should possess a photo-thermal effect (Figure S2). When GDY nanosheets were irradiated with an 808 nm laser, the temperature increased dramatically (Figure 2a). The photothermal conversion efficiency of GDY nanosheets is quite high as 42% as reported previously21. Furthermore, as shown in Figure 2b, GDY nanosheets maintained good photostability during five laser on/off cycles in water.

Figure 2. (a) Photothermal effect and (d) photostability of GDY nanosheets in water under 808 nm laser irradiation (2 W cm−2).


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Drug Loading and Release in Vitro. As a kind of 2D nanomaterial, in the same class as black phosphorus nanosheets,22-24 MoS2 nanosheets,25-26 and graphene and its derivatives,27-29 all of which have been widely reported as drug nanocarriers, GDY nanosheets possess a high surface area, suggesting they could be a novel drug delivery platform. We investigated the drug loading capacity and release behavior of GDY nanosheets by using DOX, a common chemotherapy drug in the clinic, as a model drug. By varying the DOX to GDY nanosheets feeding ratio, we achieved a loading content as high as 38% (Figure 3a,b), which exceeded recently reported exfoliated graphene nanosheets (32%).30 However, the entrapment was decreased from 75% to 30% as the DOX/GDY feeding ratio increased (Figure 3c). Considering both loading content and entrapment ratios, we chose a DOX/GDY feeding ratio of 1:1 for the following experiments.

Figure 3. (a) UV–vis-NIR absorption spectra, (d) loading content and (c) entrapment efficiency of GDY/DOX at different initial DOX/GDY feeding ratios.

GDY nanosheets not only have aromatic regions containing sp2 hybridized carbon atoms which is similar to graphene and carbon nanotubes, but also contain two acetylenic diacetylenic linkages between carbon adjacent hexagons. Due to effect of multiple conjugate bonds, the carbon-carbon single bonds between each tow diacetylenic bonds also have the properties of a carbon-carbon double bonds. This high π-conjugated property endows GDY nanosheets with a high DOX loading content through π-π stacking, considering that DOX has aromatic regions. Electrostatic interactions also contributed to the DOX loading, since the surface potential of GDY nanosheets was increased from −31.9 to +3.6 mV after loading the positively charged DOX (Figure 4a). After loading onto GDY nanosheets, the fluorescence of DOX was dramatically quenched, indicating a strong interaction between GDY nanosheets and DOX (Figure 4b). The DOX-releasing experiment was carried out at pH 5.0 and 7.4 (Figure 4c). After six hours, 10% of the DOX had been released from GDY/DOX at pH 7.4, while at pH 5.0, 15.8% of DOX had been released. The reason for this release behavior could be caused by the protonation of the daunosamine group of DOX, which increases its solubility under acidic conditions. Under laser irradiation, a higher DOX release was observed at all pH’s studied, indicating that irradiation promotes DOX release. Also, more DOX was released as the pH decreased. This is because more



daunosamine groups of DOX were protonated at the lower pH, leading to a decrease in the electrostatic interaction between DOX and the GDY nanosheets surface.

Figure 4. (a) Zeta potential of GDY nanosheets and GDY/DOX, (b) fluorescence spectra of GDY/DOX, DOX, and GDY nanosheets, (c) DOX released from GDY-DOX at pH 5.0 and 7.4 under 808 nm irradiation or not (2 W cm−2).

Antitumor Therapy in Vitro. For the in vitro treatments, first we tested the cytotoxicity of GDY nanosheets through a CCK-8 assay. As shown in Figure 5a, GDY nanosheets showed no significant cytotoxicity to MBA-MD-231, MCF-7, A549, or 16HBE cells, even when the concentration of GDY nanosheets was reached up to 100 μg/mL. Then we tested the intracellular drug release behavior by incubating GDY/DOX with MDA-MB-231 cells. Through fluorescence imaging, we can see after the first 0.5 h that there was weak red fluorescence in the cell, with GDY nanosheets largely quenching the fluorescence of DOX (Figure 5b). After 2 h, the red fluorescence became brighter and co-localized with LysoTracker-labeled lysosomes, suggesting that DOX was released from GDY nanosheets in the acid environment of the lysosome. At the end of 6 h, red fluorescence could be seen in the nucleus. Flow cytometer results also confirmed that the fluorescence intensity became higher as incubation time increased (Figure 5c). Next we tested the intracellular laser-controlled release behavior of GDY/DOX. Flow cytometer results revealed that the intracellular fluorescence intensity of DOX increased dramatically after 5 min of irradiation (Figure 5d). Clearly, the photo-dependent DOX intracellular was facilitated by the photothermal effect of GDY nanosheets.


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Figure 5. (a) Cell viability of MBA-MD-231, A549, MCF-7, and 16HBE cells with various concentrations of GDY nanosheets, (b) fluorescence images of MDA-MB-231 cells treated with GDY/DOX at different time intervals, (c) flow cytometry histogram profiles of intracellular fluorescence intensities of DOX in MDA-MB-231 cells after incubation with GDY/DOX, (d) flow cytometry histogram profiles of cellular DOX fluorescence intensities in MDA-MB-231 cells irradiated by laser or not. (scale bar = 20 μm)

To assess the photothermal therapy efficacy of GDY nanosheets in vitro, a live/dead stain assay was performed (Figure 6a). MDA-MB-231 cells were incubated with DOX, GDY nanosheets, and GDY/DOX for 4 h, with no significant cytotoxicity for any group. After irradiation for 5 min, the control and DOX groups showed few dead cells, while large numbers of dead cells were observed in the GDY nanosheets and GDY/DOX groups. This result demonstrates that GDY nanosheets can kill cancer cells directly through the hyperthermal effect. Next we used CCK-8 assay to assess the efficacy of the combined photothermal treatment and chemotherapy with GDY/DOX. We incubated MDA-MB-231 cells with GDY nanosheets and GDY/DOX at different concentrations (1.08, 2.16, or 5.4 μg/mL, equivalent to a DOX concentration of 1, 2, or 5 μM, respectively). After incubation for 4 h, MDA-MB-231 cells were rinsed with PBS, replaced with new media, and then irradiated for 5 min. Then incubation for 8 h, CCK-8 assay was performed to analyze cell proliferation. Because of low equivalent DOX



concentration and short incubation time, for the cell treated with GDY/DOX, only 20% to 24% of the cells were killed. For the GDY + laser group, along with the concentration of GDY nanosheets increasing, the cell viability was decreasing from 80% to 57%, revealing a dose-dependent PPT efficiency. As expected, the combination of photothermal- and chemotherapy enhanced the therapy efficiency in vitro. Notably, even when the concentration of GDY/DOX was as low as 5.4 μg/mL (equivalent DOX concentration 5 μM), the cell viability of combination treatment group was still notably decreased to 37%, lower than the corresponding monotherapies (Figure 6b).

Figure 6. (a) Live/dead staining assay to study the cell viability of MBA-MD-231 cells after different treatments (scale bar = 200 μm). (b) Relative viabilities of MBA-MD-231 cells under different treatments at different GDY nanosheets concentrations.

Antitumor Therapy in Vivo. For the in vivo experiments, we carried out the combination therapy using BALB/c nude tumor-bearing mice, which were separated into PBS, DOX, GDY, GDY/DOX, and their corresponding groups with irradiation. To demonstrate the photothermal activity of GDY nanosheets in the tumor, An IR thermal imaging camera was employed to monitor temperature changes of the tumor after intravenous injection with GDY/DOX under irradiation. For the GDY/DOX-treated group, the temperature of the tumors raised from ≈ 34.3 to ≈ 52.1 °C within 5 min during irradiation, which efficiently killed the tumors. By comparison, the local temperature of the tumors merely increased to 42.1 °C in the control group (Figure 7a,b). The GDY and PBS + laser groups had almost the same tumor volumes as the PBS control group, which suggests that GDY nanosheets or laser treatment alone had little therapeutic effect on tumors. Meanwhile, the DOX, DOX + laser and GDY/DOX groups all showed a better therapeutic effect because of the chemotherapeutic effect caused by DOX. Moreover, a significant inhibition of tumor growth could be found in the GDY + laser and GDY/DOX + laser groups, due to the photothermal therapeutic effect of GDY nanosheets under irradiation (Figure 7c). During the entire treatment period, no group of mice lost body weight or showed any abnormal behavior, proving that there was no acute side effect during our therapeutic process (Figure S3). All mice were euthanized and the tumors were collected and weighed at the end of the 3-week treatment. This clearly showed the excellent therapeutic efficacy of GDY/DOX to treat cancer (Figure 7d,e). To further evaluate the in vivo toxicity, the


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main organs of all the euthanized mice were dissected, weighed and subjected to histopathological examination. Compared with the normal group (mice without tumors), no treatment group demonstrated significant change. H&E staining also showed that there was no obvious damage in the major organs in any treated groups (Figure 7f and Figure S4). Moreover, serum biochemical analysis was employed to assess functional damage to major organs. The serum levels of CK, AST, ALT, TBIL, CREA, UA, and BUN of every treatment group had no significant difference from the control group, which illustrated that there were no dysfunctions of hearts, livers or kidneys (Table S1). The low systematic toxicity of GDY/DOX would be due to the low dose used in this combined therapy, compared with graphene-based platform31,32 or other 2D materials.24,33 The photothermal conversion efficiency of GDY nanosheets is quite high as 42%21 and better than Au nanorods (21%),34 bismuth sulfide nanorods (28.1%) 35 and black phosphorus quantum dots (28.4%),23 which allow us to lower the laser power and irradiation time. Therefore, our newly developed GDY drug delivery platform can be considered a safe therapeutic system.

Figure 7. (a) In vivo photothermal effect of GDY-DOX with 808 nm laser irradiation. (b) Irradiation time dependent temperature change for 808 nm irradiation. (c) tumor volume curves of tumor-bearing mice after different treatments. Group 1: PBS; Group 2: DOX; Group 3: GDY; Group 4: GDY/DOX; Group 5: PBS + laser; Group 6: DOX + laser; Group 7: GDY + laser; Group 8: GDY/DOX + laser. Tumor dissection photographs (d) and tumor weight (e) with different treatments. *P < 0.05, **P < 0.01, significant difference compared to PBS group; ##P < 0.01, significant difference compared to corresponding unirradiated groups. (f) H&E images of the heart, liver, spleen, lung, and kidney tissue of mice after the treatments (on day 22).

CONCLUSION In summary, we have demonstrated a robust multimodal therapeutic system based on GDY nanosheets. As a new kind of 2D carbon material, GDY nanosheets have a good photothermal effect and good radical-scavenging activity due to their sp hybridized



acetylene units. The GDY-based drug delivery system, using DOX as a model drug, exhibited pH-/photo- dual responsive release behavior in which DOX release was able to be promoted notably through irradiation with an NIR laser. Both cell and animal model experiments demonstrated the biosafety and enhanced antitumor effects of the photothermal/chemotherapy combination with this platform. The GDY-based drug delivery system is photostable and biocompatible, and is potentially promising for cancer theranostics. ASSOCIATED CONTENT *S Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: . Characterization data, including XPS survey spectrum of GDY nanosheets, UV–vis– NIR absorbance spectra of GDY nanosheets with various concentrations; Body weight of nude mice taken every third day after various; treatments and H&E staining of heart, liver, spleen, lung, and kidney tissue slices for different groups after treatments. (PDF) AUTHOR INFORMATION Corresponding Authors * Email: [email protected] * Email: [email protected]

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This study was supported by the National Nature Science Foundation of China (21790051), the National Key Research and Development Project of China (2016YFA0200104, 2016YFA0201600), the Science Fund for Creative Research Groups of the National Natural Science Foundation of China (11621505), CAS Key Research Program for Frontier Sciences (QYZDJ-SSW-SLH022), CAS interdisciplinary innovation team and the National Science Fund for Distinguished Young Scholars (11425520).

REFERENCES (1) Siegel, R. L.; Miller, K. D.; Jemal, A. Cancer Statistics, 2017. Ca-cancer. J. Clin. 2017, 67, 7-30. (2) Pelaz, B.; Alexiou, C.; Alvarez -Puebla, R. A.; Alves, F.; Andrews, A. M.; Ashraf, S.; Balogh, L. P.; Ballerini, L.; Bestetti, A.; Brendel, C.; Bosi, S.; Carril, M.; Chan, W. C. W.; Chen, C.; Chen, X.; Chen, X.; Cheng, Z.; Cui, D.; Du, J.; Dullin, C.; Escudero, A.; Feliu, N.; Gao, M.; George, M.; Gogotsi, Y.; Grunweller, A.; Gu, Z.; Halas, N. J.; Hampp, N.; Hartmann, R. K.; Hersam, M. C.; Hunziker, P.; Jian, J.; Jiang, X.;


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